Dentin matrix protein (DMP1) is an acidic phosphorylated protein that is an important component of the mineralized extracellular matrix of tooth and bone. Spatial-temporal expression patterns of DMP1 are highly restricted to mineralized tissues. Understanding the processes that regulate tissue-specific gene expression is an important fundamental issue in developmental biology. Currently, very little is known regarding the regulation of DMP1 expression during bone and tooth development. The purpose of this R03 proposal is to investigate through in vitro promoter studies the regulatory mechanisms of DMP1 gene expression in the highly specialized mineralized cells of bones and teeth versus other non-expressing, non-mineralizing cell types. Our preliminary studies have identified a novel element in the DMP1 promoter that potentially controls DMP1 gene expression in mineralizing versus non-mineralizing cells. DNA database analysis reveals that similar DNA consensus sequences are found in the promoters of a number of other dentin/bone genes. These in vitro studies will provide initial information to further define this novel cis-element and its function related to gene regulation. Our hypothesis is that the DMP1 gene expression in mineralizing versus non-mineralizing cells is regulated in part by a novel conserved cis-element through discrete interactions with corresponding transcription factors. In order to achieve this goal, two Specific Aims are proposed: 1) To determine consensus sequence(s) of the DNA-protein binding site by using selected and amplified binding and mutagenesis methods by electrophoretic mobility shift assay; 2) To determine the biological role of the consensus sequence(s) regulating DMP1 gene expression in the cell-specific manner by examining the cis/trans interactions using in vitro promoter-reporter analysis. Our proposed work is innovative because we have identified a novel DNA-protein binding element in DMP1 gene promoter that is conserved in the promoters of other dentin/tooth collagenous and non-collagenous genes. Results of these studies are expected to provide the foundation for in vivo studies investigating the molecular mechanisms of spatial-temporal expression patterns of DMP1 in bones and teeth as well as mineralizing and non-mineralizing tissues. These experiments will also provide significant new knowledge concerning the basic mechanism of mineralized tissue gene regulation. These studies will therefore make an important contribution toward an overall understanding of the molecular basis of bone/tooth specific gene expression.